1. Field of the Invention
This invention pertains generally to time-resolved fluorescence imaging, and more particularly to time-resolved fluorescence imaging without lifetime fitting.
2. Description of Related Art
To date, time-resolved fluorescence images are obtained by getting the lifetime pattern of the sample. Recent work in time-resolved fluorescence imaging (TRFI) has focused largely on efforts for developing mathematical algorithms to precisely extract the fluorescence lifetime from the fluorescence decay signals. After data of the fluorescence signal are acquired, the fluorescence data are analyzed and fluorescence lifetimes are estimated by fitting a decay model to the measured data. Time-resolved fluorescence data are usually complicated and are difficult to be graphically analyzed. Since the 1970s, researchers have proposed many methods and algorithms to analyze them. Today, nonlinear least squares (NLLS) is one of the most popular methods in fitting and analyzing biomedical data. Its concept is that a model starts with initial parameters and estimates the fluorescence lifetime by using iteration convolution to adjust the initial parameters and to find the best match between the measured data and the calculated data.
The time-resolved fluorescence signal of a fluorophore is usually a mono-exponential curve. However, since there is usually more than one kind of fluorophores in the specimen, the intensity decay curve is usually a combination of several exponential decay curves, which can be shown in Eq. 1:
                    I        =                              ∑            n                                                          ⁢                                    a              n                        ·                          e                                                -                  t                                                  τ                  n                                                                                        Eq        .                                  ⁢        1            where a is the amplitude and τ is the decay constant, or fluorescence lifetime.
Lifetime extraction of this multi-exponential curve is complicated and time-consuming. To make it more complicated, lifetime-extraction to obtain fluorescence images may not be reliable. Any fluorescence decay fitting and lifetime estimation methods have resolution limits. When two or more fluorescence lifetimes are closely spaced, NLLS becomes limited.
FIG. 1A and FIG. 1B show multi-exponential decay curves (in linear scale and logarithmic scale, respectively) composed of two fluorescence lifetimes (dashed and solid lines). The two lines almost perfectly overlap, although they are composed of two different sets of lifetimes. Thus, in order to accurately extract the lifetimes, complicated algorithms are developed, requiring long calculation times. The situation can be even worse when there is noise in the fluorescence signal (which is the general case), making lifetime-extraction more unreliable.
Accordingly, an object of the present invention is to overcome the restrictions of lifetime-extraction-based TRFI by obtaining time-resolved fluorescence images via low-computation calculations without lifetime calculation.